940 © 2021 The Linnean Society of London, Biological Journal of the Linnean Society, 2021, 134, 940–957 Biological Journal of the Linnean Society, 2021, 134, 940–957. With 4 figures. Phylogeography of the smooth greensnake, Opheodrys vernalis (Squamata: Colubridae): divergent lineages and variable demographics in a widely distributed yet enigmatic species BRIAN R. BLAIS 1, * ,†, , BRIAN E. SMITH 1 , JOHN S. PLACYK, JR 2,‡ , GARY S. CASPER 3 and GARTH M. SPELLMAN 4 1 School of Natural Sciences, Black Hills State University, 1200 University Street, Spearfish, SD 57799- 9008, USA 2 Department of Biology, University of Texas at Tyler, 3900 University Boulevard, Tyler, TX 75799, USA 3 University of Wisconsin-Milwaukee Field Station, 3095 Blue Goose Road, Saukville, WI 53080, USA 4 Department of Zoology, Denver Museum of Nature & Science, 2001 Colorado Boulevard, Denver, CO 80205, USA Received 1 May 2021; revised 29 July 2021; accepted for publication 2 August 2021 Phylogeographic studies can uncover robust details about the population structure, demographics, and diversity of species. The smooth greensnake, Opheodrys vernalis, is a small, cryptic snake occupying mesic grassland and sparsely wooded habitats. Although O. vernalis has a wide geographical range, many metapopulations are patchy and some are declining. We used mitochondrial DNA and double digest restriction-site associated DNA sequencing to construct the first phylogeographic assessment of O. vernalis. Genomic analysis of 119 individuals (mitochondrial DNA) and a subset of another 45 smooth greensnakes (nuclear DNA; N = 3031 single nucleotide polymorphisms) strongly supports two longitudinally separated lineages, with admixture in the Great Lakes region. Post-Pleistocene secondary contact best explains admixture from populations advancing northwards. Overall, populations expressed low heterozygosity, variable inbreeding rates, and moderate to high differentiation. Disjunct populations in the Rocky Mountains and central Great Plains regions might be contracting relicts, whereas northerly populations in more continuous mesic habitats (e.g., Prairie Pothole region, southern Canada) had signals of population expansion. Broadly, conservation management efforts should be focused on local populations, because habitat connectivity may facilitate gene flow and genetic diversity. ADDITIONAL KEYWORDS: admixture – cryptic – ddRADseq – evolutionary genomics – glaciation – multilocus – Pleistocene – population genomics – reduced representation sequencing. INTRODUCTION Contemporary population structure and diversification often reflect the ecological and evolutionary forces acting on a species (Fontanella et al., 2018). Phylogeographic studies (Avise et al., 1987; Hewitt, 2001; Knowles, 2009), especially those using large volumes of multilocus sequence data (Brito & Edwards, 2009; Edwards, 2009; Cutter, 2013; Myers et al., 2013), can provide powerful evolutionary resolution with fewer individual samples (Hohenlohe et al., 2010; Lemmon & Lemmon, 2013; Andrews et al., 2016) while linking demographics, gene flow, and population-level structure (Hickerson et al., 2010; Niemiller et al., 2012; Putman & Carbone, 2014; Pyron et al., 2016). Assessment of large panels of loci across a genome can resolve discordances from studies with relatively few markers or conflicting datasets (Manthey et al., 2015; Leavitt et al., 2020). Phylogeographic studies of snakes have uncovered cryptic genetic diversity across the range of a species *Corresponding author. E-mail: opheodrys1@gmail.com † Current address: School of Natural Resources and the Environment, University of Arizona, 1064 E. Lowell Street, Tucson, AZ 85721, USA ‡ Current address: Science Division, Trinity Valley Community College, 100 Cardinal Drive, Athens, TX 75751, USA Downloaded from https://academic.oup.com/biolinnean/article/134/4/940/6378042 by guest on 02 October 2022